Thirty percent of recombinant therapeutic proteins are made in E. coli and include important vaccine components such as carrier proteins. Further, burgeoning new immunotherapies that use proteins such as single chain antibodies and virus-like particles are enjoying increasing success. To meet the production needs of these expanding areas, Scarab Genomics proposes to extend the versatility of its Clean Genome E.coli production system to include a method by which recombinant therapeutic proteins delivered into the cell periplasm can be extracted directly into the culture medium for easy purification. Combined with Scarab?s extended fermentation platform, which enables high-density growth of Clean Genome E. coli and the constant production of target protein, a simplified recombinant protein extraction method will expand Scarab?s platform and facilitate continuous production, as mandated by the FDA. The development of an extraction method will reduce production time and effort, greatly simplify purification of therapeutic proteins such as the carrier protein CRM197 and dramatically reduce total production costs. Preliminary experiments using the carrier proteins CRM197, Haemophilus protein D and Pseudomonas exoprotein A indicate that subtle changes to the production host strain combined with a proprietary extraction buffer can induce up to 90% recovery of recombinant periplasmic protein. Further, recombinant therapeutic proteins released into the medium were stable for more than two weeks further emphasizing the versatility of the system. In Phase 1, fermentation and extraction conditions for the test carrier protein CRM197 will be improved and reagents needed to optimize expression and extraction will be developed. The first aim of Phase 2 proposes to determine whether deletions of key transpeptidase enzymes that are important in the stability of the periplasm, and which have been shown to enhance the release of recombinant proteins from the periplasm, can improve extraction. Similarly, the influence of peptidoglycan-modifying enzymes expressed from the production plasmid will be examined in the context of the extraction method. The second aim will examine the expression and extraction of four target proteins in extended fermentation to test the extraction system at production scale. In addition to the carrier proteins Haemophilus protein D and Pseudomonas exoprotein A, a single chain antibody and a single-chain variable fragment will be examined. The antibody fragments will also be subjected to purification to confirm that extraction of recombinants into culture medium simplifies purification, as was evident for carrier proteins. The third aim proposes to devise a method for removing cells and cell debris from the extracted solution with the goal of providing continuous chromatographic systems with clarified material for a truly continuous process.